CN114058680A

CN114058680A - Detection of analytes by enzyme-mediated strand displacement reaction

Info

Publication number: CN114058680A
Application number: CN202110902103.7A
Authority: CN
Inventors: 艾伦·费尔南多·罗德里格斯·塞诺拉; 邢怡铭
Original assignee: Hong Kong University of Science and Technology HKUST
Current assignee: Hong Kong University of Science and Technology HKUST
Priority date: 2020-08-06
Filing date: 2021-08-06
Publication date: 2022-02-18
Anticipated expiration: 2041-08-06
Also published as: US20220042081A1; CN114058680B; US11965204B2

Abstract

The present application relates to compositions and methods for in vitro sensors of small molecules in biological and/or environmental samples using enzyme-assisted nucleic acid reactions. The methods and compositions can be used to respond to and/or transduce signals generated by sensing of signals mediated by allosteric proteins, endonucleases, and nucleic acid reactions. The method can quickly realize one-pot analysis on the target object within several minutes. The methods and compositions can be used to generate electrochemical, fluorescent, colorimetric, and/or luminescent signals, and are compatible with detection formats such as solutions and paper-based.

Description

Detection of analytes by enzyme-mediated strand displacement reaction

Cross Reference to Related Applications

Priority of the application to U.S. provisional application No.63/103,492, filed on 6/8/2020, the entire contents of which include any tables, graphics or images, is incorporated herein by reference.

Technical Field

The present application relates generally to the in vitro detection of small molecules. In particular, the present application relates to allosteric proteins, endonucleases and nucleic acid compositions and methods for detecting small molecules or metabolites.

Background

The combination of recognition and signal transduction elements is an element that constitutes a small molecule biosensor. Small molecule sensitive recognition biomolecules such as aptamers, antibodies and allosteric transcription factors (aTF) are widely used In biosensors (see Lee, K.H. et al, In Vitro Use of Cellular Synthetic Machinery for Biosensing applications, frontiers In pharmacy. frontiers Media S.A.2019.https:// doi.org/10.3389/fphar.2019.01166). Among them, proteins such as aTF, which are transcription factors, can bind to DNA and effector molecules through different domains, binding aTF to effector molecules significantly affects the affinity for DNA. More studies have now been carried out to develop Biosensors for Detecting the corresponding cognate ligands in vivo or in vitro using such allosteric effects of natural or engineered Transcription factors (see Vanarsdale, E. et al, Redox-Based Synthetic enzymes Electrochemical Detection of the Herbicides Dicamba and Roundp via Rewired Escherichia coli 2019,4,1180-1184.https:// doi.org/10.1021/ansensors.9b00085; Wen, K.Y.865. et al, Freemont, P.S.A-Free Biosensor for Detecting Quorum Sensing Molecules in P.Aerinogena-Infed DNA research Resors.SaponySaponySaponySaponySaponySaponySaponySaponySaponySus.2017, Biotechnology J.23019, Biotech, Biotech. 31/19. J.00219. Biotech. 19. Biotech. 31/16. Biotech. 31. J.8. Biotech. Biotech. J.31. for Detecting DNA antigens, 3619. Biotech. J.8. Biotech.

Allosteric transcription factors in vitro biosensors can be generally seen as divided into two categories: 1) in a Cell-Free system, an allosteric transcription factor is used to regulate the enzymatic activity of RNA polymerase on the promoter thereof to establish a detection platform (see Silverman, A.D et al, Design and Optimization of a Cell-Free Atrazine biosensor, ACS Synth. biol.2020,9,671-677.https:// doi. org/10.1021/acssynbio. 9b00388; jewett, M.C. et al, On Demand, Portable, Cell-Free Molecular Sensing platform, WO/2020/072127, April 9,2020); 2) the detection platform is established by combining response protein and molecular signal transduction. aTF the principle of the competition of DNA interacting biomolecules (usually enzymes) for the same double stranded DNA fragment is widely used in biosensors. These methods can be summarized as follows: when the cognate ligand is present and the aTF-ligand complex dissociates from the DNA, the competing enzyme can act on the DNA and produce a product that can then be amplified. For example, T4 ligase can be used to compete with aTF for nicked double stranded DNA substrates that are nicked in the binding sequence of aTF. When aTF responds to effectors, the T4 ligase can repair the DNA and can amplify it by Polymerase Chain Reaction (PCR), Recombinase Polymerase Amplification (RPA) or Rolling Circle Amplification (RCA) (see Cao, J et al, Harnessing a previous university transduction primers for Sensing diversity microorganisms in vitro. Sci. adv.2018,4, eaau4602.https:// doi. org/10.1126/sciadv. aaau4602). Competition of the aTF HosA with Klenow Fragment (KF) has been used to detect p-hydroxybenzoic acid (PHBA). KF simultaneously initiates a Strand Displacement Amplification (SDA) cycle with Nb.BbvCI (a nicking endonuclease) when PHBA is present, wherein the product of SDA folds into a G-quadruplex that produces a fluorescent or colorimetric signal (see Yao, Y. et al, Development of Small molecular Biosensors by Coupling the Recognition of the Bacterial exogenous Transcription Factor with Isothermal amplification and discrimination amplification. chem.Commun.2018,54,4774-4777. https:// doi.org/10.1039/c8cc01764 f). Restriction endonuclease HindIII is used to cleave sequences that bind HucR to DNA in the presence of Uric Acid, where there is a negative correlation between Uric Acid concentration and Ct value of qPCR (Yao, Y. et al, Novel Signal Transduction System for Development of Uric Acid biosensors. appl. Microbiol. Biotechnol.2018,102, 7489-7497. https:// doi. org/10.1007/s 00253-018-. Recently, researchers developed a Cas12a Platform for the Detection of uric acid (see Liang, M. et al, A CRISPR-Cas12a-Derived Biosensing Platform for the high Sensitive Detection of variant Small molecules, Nat. Commun.2019,10.https:// doi. org/10.1038/s 41467-019-. aTF-ligand complex formation enables Ribonucleoprotein (RNP) to bind to and cleave target DNA, thereby activating the trans-cleavage activity of Cas12a to generate a fluorescent signal.

Currently, the methods developed suffer from a number of disadvantages. Most of the current methods require multiple preparation steps including culturing, equilibration, washing, centrifugation and the like, which not only prolong the total time from the preparation of the reaction to the acquisition of the detection result, but also make the experimental system more complicated. Meanwhile, the signal response and amplification need to be performed step by step, so that the detection cannot be realized on the same container or carrier. Some immobilization-based aTF detection platforms tend to be limited by the methods and efficiencies of molecular immobilization and modification. Thus, many biosensors still suffer from long turnaround times (> 100min), the need for complex formulations and chemical modifications, or low sensitivity or instability. Furthermore, most current techniques still rely on nucleic acid amplification, which adds virtually to the cost of the assay and makes the reaction more complex.

Therefore, there is still a need to develop a platform that can rapidly and accurately detect small molecules without the above-mentioned drawbacks.

Disclosure of Invention

The present application relates to compositions and methods for in vitro sensors for the detection of small molecules in biological and/or environmental samples using enzyme-assisted nucleic acid reactions. The compositions and methods can be used to respond to and/or transduce sensory signals mediated by allosteric proteins or aptamers, endonucleases, and nucleic acid reactions. The compositions and methods can be used to generate electrochemical, fluorescent, colorimetric, and/or luminescent signals, and the methods can be performed in different modes, such as, for example, in solution, paper-based, and modes that utilize microfluidic devices. The methods, compositions and compositions can be modified to accommodate different preservation techniques, such as lyophilization and drying on paper.

The methods include molecular mechanisms, assay devices, reaction components and compositions, and methods of generating and measuring signals. Typically, the test sample may be prepared and processed prior to detection. Preparation may include filtration, centrifugation, temperature change, solubilization, dilution, or concentration of the target molecule in the test sample. The molecular mechanism and experimental setup is capable of sensing specific target small molecules/metabolites mediated by allosteric proteins or aptamers in the test sample. If the target small molecule/metabolite is present in the test sample, the target small molecule/metabolite can bind to the allosteric protein or aptamer and trigger the cycle of the Toehold-mediated strand displacement reaction initiated by the endonuclease. The final displacement products accumulated in the reaction can be detected by electrochemical, fluorescent, colorimetric and/or luminescent methods. The method adopts the Toehold-mediated strand displacement amplification, and only adopts competitive enzyme or the change of the DNA template structure to trigger in the signal amplification process, so that the nucleic acid amplification process in the existing method is avoided, and the rapid, simple and accurate one-step analysis can be realized. The method can be applied to virtually any allosteric protein/ligand complex or aptamer that can act as a repressor or activator.

The assay device and the manner of reaction are compatible with different forms and variations of various biosensing strategies, while the components and compositions of the reaction are also applicable to immobilization-based detection platforms.

Drawings

Figure 1 is a schematic representation of an in vitro biosensor for small molecules/metabolites. The natural or engineered allosteric protein (aTF) binds to a double-stranded DNA fragment that includes its cognate DNA binding sequence. Binding of this protein prevents type IIS restriction endonucleases (HgaI in this case) from cleaving the DNA binding sequence, with the recognition site of the type IIS restriction endonuclease being located either upstream or downstream of the DNA binding sequence. The cleavage site of the type IIS restriction endonuclease is located in the allosteric protein binding sequence. The presence of a cognate effector or ligand (e.g., a small molecule or metabolite) of the allosteric protein causes the allosteric protein to dissociate from the binding DNA sequence, which allows HgaI to bind to its recognition site and cleave the allosteric protein-binding DNA sequence. This cleavage produces a 5' overhang (short portion of single-stranded DNA) of five nucleotides in length. For the Invasive Probe (IP), the overhang serves as a Toehold region and nucleation site, initiating a Toehold-mediated strand displacement reaction. The IP is labeled at the 5 'end and 3' end with a fluorophore and a quencher group, respectively. The result of this reaction is an intermediate (S-IP) that can be cleaved by HgaI, but lacks the complete binding sequence for allosteric proteins. This cleavage creates a region of Toehold that initiates another Toehold-mediated strand displacement reaction and causes displacement of the IP (P) fragment, thereby generating a fluorescent signal. The novel S-IP participates in a cyclic reaction including cleavage by type IIS restriction enzymes and Toehold-mediated strand displacement. The total amount of IP decreases and P increases throughout the cycle. Each cycle allows the displaced fluorescent product (P) to accumulate continuously and the fluorescence signal can be measured in real time or at an end point.

Fig. 2 shows a diagram of a separate signal amplification loop. The circuit has three main parts: type IIS restriction enzyme HgaI, DNA intermediate (S-IP) and Invasion Probe (IP).

FIG. 3 shows the demonstration that the signal amplification loop is dependent on the concentration of DNA template (S). The final volume of the reaction was 10 μ L and consisted of: 1.25 μ M IP, 100U/mL HgaI and different concentrations of DNA template (S) (50nM, 75nM and 100 nM).

Fig. 4 shows a demonstration that the signal amplification loop depends on the Invasive Probe (IP) concentration. The final volume of the reaction was 10 μ L and consisted of: 50nM DNA template (S), 100U/mL HgaI and different concentrations of Invader Probe (IP) (50nM, 125nM, 250nM, 500nM and 1250 nM).

Fig. 5A to 5C show that allosteric effect mediates HgaI cleavage. FIG. 5A shows an Electrophoretic Mobility Shift Assay (EMSA) for confirming binding of TetR to a DNA template (S) containing an HgaI recognition site and a tetO sequence; and unbound in the presence of tetracycline (Tc). Fig. 5B shows a PAGE of the effect of a 2-bp spacer (N ═ 0-12) on HgaI cleavage of TetR-protected DNA. 50nM of DNA was incubated with 250nM of TetR and 1U of HgaI at 37 ℃ for 30 min. Immediately thereafter, the protein was digested with 0.4U of proteinase K at 37 ℃ for 15 minutes. The rectangle indicates the cleavage product of S. The arrow indicates the TetR/S complex, since TetR is not completely digested by proteinase K. Fig. 5C shows EMSA with Tc modulating HgaI cleavage. The increased Tc concentration gives a greater amount of cleaved product.

Fig. 6A to 6C show the experiment of the biosensor using the environmental substrate. Fig. 6A shows environmental samples taken at a hong kong pond. After collection, the samples were spiked with different antibiotics (where applicable) and immediately filtered using syringe filters. Fig. 6B shows that the entire workflow from sample filtration to result can be completed in 25 minutes. Figure 6C shows that the sensor successfully detected the presence of incorporated antibiotics in the water sample and that no interference occurred. 500nM of each ligand was used in a TetR-based biosensor (final concentration 250 nM). 2.5 μ M of each ligand was used for the MphR-based biosensor (final concentration 1.25 μ M). (ns: p value > 0.05 between non-homologous ligand and WS using two-tailed t-test). Tc ═ tetracycline, Ery ═ erythromycin, Amp ═ ampicillin, Kan ═ kanamycin, WS ═ water samples (no analyte). Bars represent mean values and error bars represent s.d.n.2 (fig. 6C).

FIGS. 7A-7B show the demonstration that the endonuclease-mediated Toehold-mediated strand displacement (TMSD) reaction can be negatively regulated (repressed) by TetR and de-inhibited by tetracycline (Tc). Fig. 7A shows portions of a biosensor, indicating DNA template (S) and Invasion Probe (IP) sequences and modifications. FIG. 7B shows the inhibition of the response using different concentrations of TetR (0nM, 50nM, 100nM, 150nM and 200 nM).

FIG. 8 shows the demonstration that TetR-based biosensors responded to different concentrations of tetracycline (0nM, 25nM, 50nM, 100nM, 250nM and 500 nM).

FIGS. 9A-9B show the demonstration that the endonuclease-mediated Toehold-mediated strand displacement (TMSD) reaction can be negatively regulated (repressed) by MphR and de-inhibited by erythromycin (Ery). Fig. 9A shows portions of a biosensor, indicating DNA template (S) and Invasion Probe (IP) sequences and modifications. FIG. 9B shows inhibition of the response using different concentrations of MphR (0nM, 750nM, 1.5. mu.M, 2.25. mu.M and 3. mu.M).

FIG. 10 shows that the MphR-based biosensor responded to different concentrations of erythromycin (0nM, 100nM, 200nM, 350nM, 500nM, 750nM, 1. mu.M, and 2.5. mu.M).

FIGS. 11A-11B are graphical representations showing that endonuclease-mediated Toehold-mediated strand displacement (TMSD) reactions can be used to transduce and amplify signals in aptamer sensors. Fig. 11A shows structural changes when DNA template S is hybridized to an Invasive Probe (IP) after target-induced aptamer dissociation. Similarly, fig. 11B shows a structural change of the aptamer switch composed of an aptamer domain and an S domain. After the structural change, both mechanisms can participate in the signal amplification loop.

Fig. 12A to 12B show that a signal amplification loop can be triggered by a conformational change on the DNA template S. Fig. 12A shows the structure and Minimum Free Energy (MFE) of two different hairpins (SH and LH) from S. FIG. 12B shows that the length of the hairpin stem has an effect on the velocity and leakage (rate and leakage) of the signal amplification loop, and the final concentration of cleaved fluorescent product. SH and LH are systems without aptamers, while SH suppression and LH suppression are systems with aptamers at equimolar concentrations to S. In all cases: the reaction volume was 10. mu.L, with HgaI at a concentration of 100U/mL, S at a concentration of 50nM, and IP at a concentration of 1.25. mu.M.

FIG. 13 shows a graphical representation of a Lateral Flow Assay (Lateral Flow Assay) format. The left panel shows the components of the test strip and the modification of the invader probe. The middle panel shows a simplified schematic of the negative test. The right hand side shows a simplified schematic of a positive test.

FIG. 14 shows a schematic representation of a detection electrochemical signal amplification circuit.

FIG. 15 shows SDS-PAGE of purified TetR and MphR in the reduced state.

Brief description of the sequences

1, SEQ ID NO: nucleotide sequence of E.coli (E.coli) encoding TetR

2, SEQ ID NO: exemplary DNA template sequences for use with TetR sensor molecules

3, SEQ ID NO: complementary sequence of SEQ ID NO 2

4, SEQ ID NO: exemplary invasive Probe

5, SEQ ID NO: exemplary DNA template sequences for use with TetR sensor molecules

6 of SEQ ID NO: complementary sequence of SEQ ID NO 5

7, SEQ ID NO: exemplary DNA template sequences for use with TetR sensor molecules

8, SEQ ID NO: complementary sequence of SEQ ID NO 7

9 of SEQ ID NO: exemplary DNA template sequences for use with TetR sensor molecules

10, SEQ ID NO: complementary sequence of SEQ ID NO 9

11, SEQ ID NO: exemplary DNA template sequences for use with TetR sensor molecules

12, SEQ ID NO: complementary sequence of SEQ ID NO 11

13 in SEQ ID NO: exemplary DNA template sequences for use with TetR sensor molecules

14, SEQ ID NO: complementary sequence of SEQ ID NO 13

15, SEQ ID NO: exemplary DNA template sequences for use with TetR sensor molecules

16 in SEQ ID NO: complementary sequence of SEQ ID NO. 15

17 in SEQ ID NO: exemplary DNA template sequences for use with MphR sensor molecules

18, SEQ ID NO: complementary sequence of SEQ ID NO 17

19, SEQ ID NO: exemplary invasive Probe

20, SEQ ID NO: exemplary invasive Probe

21, SEQ ID NO: amino acid sequence of TetR E.coli

22, SEQ ID NO: amino acid sequence of MphR Escherichia coli

Detailed Description

Selected definition

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, when the terms "including, includes", "having", "with", or variants thereof are used in the detailed description and/or claims, such terms are intended to be inclusive in a manner similar to the term "comprising". Transitional terms/phrases (and any grammatical variants thereof) "comprising" includes the phrases "consisting essentially of … … (consistency of consistency)" and "consisting of … … (consistency )".

The phrase "consisting essentially of … …" means that the claims include embodiments that include the specified materials or steps as well as embodiments that do not materially affect the basic and novel characteristics of the claims.

The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Where particular values are described in the application and claims, unless otherwise stated, the term "about" shall be assumed to mean within an acceptable error range for the particular value.

In the present disclosure, ranges are set forth in shorthand form to avoid having to set forth and describe each and every value within a range in detail. Any suitable value within the range can be selected as the upper, lower, or end value of the range, where appropriate. For example, a range of 1 to 10 indicates end values of 1 and 10, and intermediate values of 2, 3,4, 5, 6, 7, 8,9, and all intermediate ranges included within 1 to 10, such as 2 to5, 2 to 8, and 7 to 10. Likewise, when ranges are used herein, it is intended to expressly include combinations and sub-combinations of ranges (e.g., sub-ranges within the disclosed ranges) and specific embodiments therein.

As used herein and in the claims, "sample" refers to a sample of cells, tissue, solids, or fluids, including but not limited to, for example, skin, plasma, serum, spinal fluid, lymph, synovial fluid, urine, tears, blood cells, organs, tumors, environmental sources (including water courses, soil, or air), in vitro cell culture components (including but not limited to conditioned medium, recombinant cells, and cell components resulting from the growth of cells in cell culture medium), or samples derived from an organism or any other source containing an organism.

The term "organism" as used herein includes viruses, bacteria, fungi, plants and animals. Other examples of organisms are known to those of ordinary skill in the art, and such embodiments are within the scope of the materials and methods disclosed herein. The assays described herein can be used to analyze any genetic material obtained from any organism.

As used herein, a "template" or "template sequence" is a polynucleotide (e.g., as defined herein, including DNA, RNA, or DNA/RNA hybrids, and modified forms thereof) that includes a "target site". The term "target site" is used to refer to a nucleic acid sequence present in a template sequence that can bind to a probe (e.g., any of the probes herein), protein, or other nucleotide sequence, so long as sufficient binding conditions (e.g., sufficient complementarity) are present. Suitable DNA/RNA binding conditions include physiological conditions normally present in a cell. Other suitable binding conditions (e.g., conditions in a cell-free system) are known in the art.

The term "hybridize" when used with respect to two sequences means that the two sequences are sufficiently complementary to each other to enable base pairing of nucleotides between the two sequences. Sequences that hybridize to each other may be perfectly complementary, but may also have some degree of mismatch. Thus, a probe sequence or other hybridizing sequence may have some mismatches with a corresponding target sequence, but still function as intended, so long as the probe or other hybridizing sequence can hybridize to the target sequence. Depending on the stringency of hybridization, up to about 5% to 20% mismatch between two complementary sequences may allow hybridization between the two sequences. Generally, high stringency conditions have higher temperatures and lower salt concentrations, while low stringency conditions have lower temperatures and higher salt concentrations. High stringency conditions are preferred for hybridization. It is indicated with respect to two sequences that the two sequences are sufficiently complementary to each other to enable base pairing of nucleotides between the two sequences.

"hybridization conditions" refers to conditions of temperature, pH, and reactant concentration that enable at least a portion of complementary sequences to recombine with each other. The conditions required to accomplish hybridization depend on the size of the oligonucleotides to be hybridized, the degree of complementarity between the oligonucleotides, and the presence of other substances in the hybridization reaction mixture. The actual conditions required for each hybridization step are well known in the art or can be readily determined by one of ordinary skill in the art. Typical hybridization conditions include the use of a pH of about 7 to about 8.5 and a temperature of about 30 ℃ to about 80 ℃; preferred conditions include the use of Tris-EDTA buffer at pH 7.5 and from about 5mM to about 15mM MgCl₂. The mixed oligomer was heated to 95 ℃ and slowly cooled to 20 ℃ over 180 minutes. In certain embodiments, the temperature of hybridization must be below the melting temperature of the DNA duplex. Hybridization conditions may also include buffers that are compatible, i.e., chemically inert, with respect to the oligonucleotides and other components, but still allow hybridization between complementary base pairs.

As used herein, the phrases "connectable" or "associable with" used interchangeably refer to a juxtaposition in which the components are in a relationship that allows them to function in their intended manner. The first component may be capable of being linked to the second component by any useful bond (e.g., a covalent bond, a non-covalent bond, and/or a bond linked by van der waals forces, hydrogen bonds, and/or other intermolecular forces such as those involving pi-pi interactions, salt bridges, or cation-pi interactions) or any useful linker (e.g., a nucleotide sequence or any sequence herein).

Throughout this disclosure, different sequences, such as target sequences and probe sequences, are described by specific nomenclature. When such nomenclature is used, it is understood that the identified sequence is substantially identical to or substantially reverse complementary to at least a portion of the corresponding sequence. For example, an "invasive probe sequence" describes a sequence that is substantially identical to at least a portion of an invasive probe sequence or is substantially reverse complementary to at least a portion of an invasive probe sequence. Thus, this nomenclature is used herein to simplify the description of the different polynucleotides and portions of polynucleotides used in the methods disclosed herein; however, one of ordinary skill in the art will recognize that appropriate sequences that are substantially identical or substantially reverse complementary to at least a portion of the corresponding sequence may be used to practice the methods disclosed herein.

Furthermore, two sequences corresponding to each other, such as a probe-binding sequence, a DNA template sequence or an aptamer sequence, have at least 90% sequence identity, preferably at least 95% sequence identity, even more preferably at least 97% sequence identity and most preferably at least 99% sequence identity over at least 70%, preferably at least 80%, even more preferably at least 90% and most preferably at least 95% of the sequences. Alternatively, two sequences corresponding to each other are reverse complementary to each other and have at least 90% perfect match, preferably at least 95% perfect match, even more preferably at least 97% perfect match, and most preferably at least 99% perfect match over at least 70%, preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the sequences in the reverse complementary sequences. Thus, two sequences corresponding to each other may hybridize to each other or to a common reference sequence over at least 70%, preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the sequences. Preferably, two sequences corresponding to each other are 100% identical over the entire length of the two sequences, or 100% reverse complementary over the entire length of the two sequences.

As used herein, the phrase "allosteric protein" refers to a protein that binds directly to the recognition and response of a molecule of interest. Allosteric effects are a common feature of proteins, in which the behavior of the "active" site is altered by binding of an effector to a second or "allosteric" site (usually quite distant from the first site). The altered behavior may directly or indirectly cause a change in the activity of the protein, thereby causing a detectable response.

As used herein, the term "Toehold" refers to a region comprising the starting site of a nucleic acid sequence designed to initiate hybridization of the region to a complementary nucleic acid sequence. The secondary structure of the nucleic acid sequence may be in the form of a Toehold exposure or inactivation. For example, in some embodiments, the secondary structure of the Toehold renders the Toehold available for hybridization to complementary nucleic acids (the Toehold is "exposed" or "accessible"), and in other embodiments, the secondary structure of the Toehold renders the Toehold unavailable for hybridization to complementary nucleic acids (the Toehold is "inactivated" or "inaccessible"). If Toehold is inactivated or otherwise unavailable, Toehold can be made available by some signal, such as the opening of a hairpin, which is a sequence or portion thereof cleaved by a restriction endonuclease. When exposed, the Toehold is configured such that complementary nucleic acid sequences can nucleate at the Toehold.

As used herein, an "aptamer" is an oligonucleotide that is capable of specifically binding to a target analyte in addition to hybridizing by base pairing. Aptamers typically comprise DNA or RNA or a mixture of DNA and RNA, but may also contain proteins in addition to nucleotides, or proteins without nucleotides. Aptamers can be naturally occurring or prepared by synthetic or recombinant methods. Aptamers are typically single-stranded, but can also be double-stranded or triple-stranded. Aptamers may comprise naturally occurring nucleotides, nucleotides modified in some way, for example by chemical modification, and non-natural bases. Aptamers may contain certain chemical modifications, for example by the addition of a label such as a fluorescent group, or by the addition of a molecule that enables the aptamer to be cross-linked to the molecule to which it is bound. Aptamers are the same "type" if they have the same sequence or are capable of specifically binding to the same molecule. Aptamers will vary in length, but are typically less than about 100 nucleotides in length.

As used herein, the term "cleavage" refers to the breaking of the covalent backbone of a nucleic acid molecule. Cleavage can be initiated by a variety of methods, including but not limited to enzymatic or chemical hydrolysis of the phosphodiester bond. Both single-stranded and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two different single-stranded cleavages. DNA cleavage can produce blunt ends or staggered ends.

As used herein, the terms "nuclease" and "endonuclease" and the phrase "restriction enzyme" are used interchangeably herein to refer to an enzyme that has DNA cleavage catalytic activity.

If one component of a substance is present in a higher molar concentration than another component, it is generally said that the substance is present in "excess" or "molar excess" relative to the other component. Typically, when present in excess, this component will be present in at least about a 2-fold, at least about a 5-fold, or at least about a 10-fold molar excess, and typically in a 100-to 1,000,000-fold molar excess. One skilled in the art will appreciate and understand the particular degree or amount of excess that is preferred for any particular reaction or reaction condition. Such excess is generally determined empirically and/or is optimal for a particular reaction or reaction conditions.

The present disclosure provides materials and methods that address problems associated with conventional methods for detecting small molecules, while speeding up the process.

Certain embodiments of the present application relate to a method of determining the presence of an analyte in a sample, the method comprising a) bringing into mixed contact with the sample an endonuclease, a DNA template, a sensor molecule, and an invasive probe, wherein the sensor molecule can inhibit recognition of a restriction enzyme site in the DNA template by the restriction endonuclease; b) binding or hybridizing a sensor molecule to the DNA template, wherein the sensor molecule prevents the restriction enzyme from recognizing a restriction enzyme site in the DNA template specific for the restriction enzyme when the analyte is not present, or displaces the sensor molecule from the DNA template when the analyte binds to the sensor molecule; c) optionally, digesting the DNA template with a restriction enzyme; d) optionally, hybridizing an invader probe to the DNA template; e) optionally, cleaving the hybridized invader probe and DNA template with a restriction enzyme; and f) determining the presence or absence of at least one analyte by detecting a signal released from the invasive probe, wherein detection of the signal is indicative of the presence of the analyte. Certain embodiments also provide a composition for the detection of an analyte, the composition comprising a restriction enzyme, a DNA template, a sensor molecule, and an invasive probe, wherein the analyte binds to the sensor molecule and allows recognition of a restriction enzyme site in the DNA template by the restriction enzyme. In certain embodiments, the invader probe is associated with a reporter molecule to generate a detectable signal using a Toehold-mediated strand displacement. In certain embodiments, the sample may be treated by heating, centrifugation, chemical or physical dissolution, dilution, concentration, or filtration prior to contact with the reaction mixture.

DNA template and Toehold mediated Strand Displacement

The methods of the present application are capable of detecting a variety of target molecules. In one aspect, the present application relates to compositions and methods using sensor molecules, such as allosteric protein, transcription factor, aptamer, or aptamer sensors, that bind to and are capable of detecting a target molecule, wherein the sensor inhibits the binding and cleavage of a DNA template by an endonuclease.

In certain embodiments, the DNA template may be single-stranded or double-stranded. In certain embodiments, the DNA template may have at least one, two, three, four, five, six, seven, eight, or more sequences that facilitate processing or detecting the analyte. Such sequences include restriction or endonuclease sites, particularly type IIS endonuclease sites, spacer sequences of about 1 to about 20 base pairs, and/or at least one, two, three, four, five, six, seven, eight or more sites complementary to aptamers. In certain embodiments, the DNA template may have an operator sequence, which is preferably a DNA binding sequence of the sensor molecule. In certain embodiments, the sequence comprising the operator sequence, the restriction enzyme site, and the sequence complementary to the aptamer is ligatable. In certain embodiments where there are two or more sites that facilitate processing or detection of the analyte (e.g., restriction enzyme sites, operator sequences, or aptamer annotated regions), these sites can be separated by zero base pairs or at least 1,2, 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more base pairs. In preferred embodiments where there are two or more sites (e.g., restriction enzyme sites, operator sequences, or aptamer annotated regions) that facilitate processing or detection of the analyte, these sites may be separated by about 4 to about 15 base pairs.

In certain embodiments, the binding and cleavage of a restriction enzyme to a DNA template can allow hybridization of the probe to the cleaved DNA template and initiate a Toehold-mediated Strand Displacement, as described in U.S. patent No.9,284,602 and Simmel FC, Yurke B, single hr. principles and Applications of Nucleic Acid Strand and Displacement Reactions (principles and Applications of Nucleic Acid Strand Displacement Reactions). Chem rev.2019, 5 months 22 days; 119(10) 6326-6369; 10.1021/acs, chemrev.8b00580. electronic publication in 2019, 2/month, 4; toehold-mediated strand displacement, described in PMID:30714375, is a well-known method in the art, and each of the above references is incorporated herein by reference. In a preferred embodiment, the presence of the analyte is determined indirectly by means of a label cleaved from the probe. In certain embodiments, when the analyte is present, the sensor molecule no longer inhibits the restriction enzyme from recognizing a restriction enzyme site in the DNA template, and the DNA template is cleaved. The cleaved DNA template may then be hybridized to a probe, which may have a fluorescent label and a quenching group, or an electrochemical label, capable of ligation. In certain embodiments, the DNA template hybridized to the probe may be cleaved by one or more restriction endonucleases. This cleavage can remove the quenching group or electroactive label from the probe, thereby enabling detection of the analyte and opening of the Toehold DNA strand and creating an overhang of at least one, two, three, four, five, six, seven, eight, nine, ten, or more nucleotides in length on the unmodified DNA template strand that serves as a Toehold region and nucleation site for another invader probe to initiate a Toehold-mediated strand displacement reaction (TMSD). The result of this reaction is the displacement and accumulation of the label, preferably the displacement and accumulation of a fragment of the invasive probe containing a fluorophore or an electroactive label. The newly formed hybrid invasive probe and DNA template participate in a cycling reaction involving cleavage by one or more restriction enzymes followed by TMSD.

In certain embodiments, hybridization of a probe (particularly an invasive probe) to a nucleotide sequence (particularly a DNA template) can be achieved by any method that allows the probe to recombine with the DNA template. In one embodiment, hybridization can be achieved by means of a Toehold-mediated strand displacement. Hybridization can be triggered by recombination of the probe with a Toehold sequence on a partially double-stranded polynucleotide strand. Recombination of the Toehold sequence on a partially double-stranded polynucleotide strand with a probe may result in strand separation of the partially double-stranded polynucleotide strand and hybridization of the probe to the Toehold-containing polynucleotide sequence. The strand not containing the Toehold sequence was subjected to substitution. It will generally be appreciated that the specificity of the hybridization reaction of a probe with a polynucleotide sequence may be thermodynamically controlled by the probe sequence and/or the length of the Toehold region.

Cleavage of the hybridized probe and polynucleotide sequence will cause a signal to be released from the probe. Detection of the presence of the released signal may indicate the presence of the analyte in the sample. The strength of the signal may be measured relative to a reference signal. The reference signal may be a signal from a sample with a known analyte. The reference signal may also be a signal taken prior to addition of the enzyme (i.e.the restriction enzyme may be added to the sample after the DNA template, the sensor molecule and the appropriate reaction components) or prior to removal or replacement of one or more probes. The reference signal can also be a signal collected from a sample in the absence of hybridization of the probe to the polynucleotide sequence.

In certain embodiments, the DNA template sequence may have a nucleotide spacer sequence between the recognition site of the sensor molecule and the restriction enzyme site, which means that the present application can be used with almost any aTF or aptamer without requiring overlapping sequences between the recognition site of the endonuclease and the operator sequence, such as the DNA binding sequence of aTF. In certain embodiments, the recognition site of the sensor molecule may be separated from the restriction enzyme cleavage site by 1,2, 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases.

Probe design and detection

In certain embodiments, the probe may be designed to hybridize to a template DNA sequence or portion thereof. In certain embodiments, the complementary nucleotide segment of the probe is 1,2, 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or 100 base pairs in length or longer. In a preferred embodiment, the complementary nucleotide segment of the probe is from about 10 to about 50 base pairs in length. In addition, a probe (e.g., any of the probes herein, such as an invasive probe) can be labeled with a fluorescent label (e.g., used with a quencher label), an electroactive label, or can be unlabeled. The probe may have an endonuclease binding site. The concentration of the probe can be optimized to facilitate the amplification reaction.

In certain embodiments, the probes herein can include any useful label, including fluorescent labels and quencher labels at any useful position in the nucleic acid sequence, e.g., at the 3 'end and/or 5' end of the probe or within a loop structure. Exemplary fluorescent labels include quantum dots or fluorophores. Examples of fluorescent labels for use in the method include fluorescein, 6-FAM^TM(Applied Biosystems, Calsbad, Calif.), TET^TM(Applied Biosystems, Calsbad, Calif.), VIC^TM(Applied Biosystems, Calsbad, Calif.), MAX, HEX^TM(Applied Biosystems, Calsbad, Calif.), TYE^TM(ThermoFisher Scientific, Waltherm, Mass.), TYE665, TYE705, TEX, JOE, Cy^TM(Amersham Biosciences of Piscataway, N.J.) dyes (Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7), Texas

(Molecular Probes, Inc. of Yougu, Oregon), Texas Red-X,

(Molecular Probes, Inc. of Yougu, Oregon.) dye (AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 532, AlexaFluor 546, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750), DyLight^TM(ThermoFisher Scientific, Waltherm, Mass.) dyes (DyLight 350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, Va,DyLight 649、DyLight 755)、ATTO^TM(ATTO-TEC GmbH of stannum germany) dyes (ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 520, ATTO 532, ATTO 550, ATTO 565, ATTO Rhol01, ATTO 590, ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 635, ATTO 637, ATTO 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740)

(Molecular Probes, Inc. of Yougu, Oregon.) dye (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), HiLyte Fluor (Anaspec of Simon, Calif.) dye (HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 594, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750), AMCA-S,

Blue (Molecular Probes, Inc. of Yougu, Oregon), Cascade Yellow, coumarin, hydroxycoumarin, Rhodamine Green^TM-X (Molecular Probes, Inc. of Ewing, Oregon), Rhodamine Red^TM-X (Molecular Probes, Inc. of Ewing, Oregon), Rhodamine 6G, TMR, TAMRA^TM(Applied Biosystems, Calsbad, Calif.), 5-TAMRA, ROX^TM(Applied Biosystems, Calsbad, Calif.), Oregon

(Life Technologies, Greenland, N.Y.), Oregon Green 500, and,

700 (Li-Cor Biosciences of Lincoln, Nebraska), IRDye 800, WeIIRED D2, WeIIRED D3, WeIIRED D4, and

640 (of Mannheim, Germany)Roche Diagnostics GmbH). In some embodiments, extinction coefficients > 50,000M may be used^-1cm^-1And a bright fluorophore that is spectrally matched appropriately to the fluorescence detection channel.

In certain embodiments, a fluorescently labeled probe is included in the reaction mixture and a fluorescently labeled reaction product is produced. The fluorescent group included in embodiments of the methods and compositions of the present application that is used as a label to produce a fluorescently labeled probe can be any of a number of fluorescent groups, including but not limited to 4-acetamido-4 '-isothiocyanatostilbene-2, 2' -disulfonic acid; acridine and derivatives, such as acridine and acridine isothiocyanate; 4-amino-N- [ 3-vinylsulfonyl) phenyl]Naphthalimide-3, 5-disulfonate, fluorescein (Lucifer Yellow) VS; n- (4-anilino-1-naphthyl) maleimide; anthranilamide, Brilliant Yellow (Brilliant Yellow); BIODIPY fluorophore (4, 4-difluoro-4-boron-3 a,4 a-diaza-s-indacene); coumarins and derivatives, such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumaran 151); acid red (cyanosine); DAPDXYL sulfonyl chloride; 4', 6-diamidino-2-phenylindole (DAPI); 5',5 "-dibromopyrogallol-sulfonphthalein (Bromopyrol Red); 7-diethylamino-3- (4' -isothiocyanatophenyl) -4-methylcoumarin; diethylenetriamine pentaacetate; 4,4 '-diisothiocyanatodihydro-stilbene-2, 2' -disulphonic acid; 4,4 '-diisothiocyanatostilbene-2, 2' -disulphonic acid; 5- [ dimethylamino group]Naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-4' -dimethylaminophenylazo) benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4' -isothiocyanate (DABITC); EDANS (5- [ (2-aminoethyl) amino group)]Naphthalene-1-sulfonic acid), eosin and derivatives, such as eosin isothiocyanate; erythrosine and derivatives, such as erythrosine B and erythrosine isothiocyanate; ethidium, such as ethidium bromide; fluorescein and derivatives, such as 5-carboxyfluorescein (FAM), hexachlorofluorescein, 5- (4, 6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2',7' -dimethoxy-4 ',5' -dichloro-6-carboxyfluorescein (JOE) and Fluorescein Isothiocyanate (FITC); fluorescamine; green fluorescent proteins and derivatives, such as EBFP, EBFP2, ECFP and YFP; IAEDANS (5- ({2- [ (iodoacetyl) amino)]Ethyl } amino) naphthalene-1-sulfonic acid), malachite green isothiocyanate; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; pararosaniline; phenol red; b-phycoerythrin; o-phthalaldehyde; pyrene and derivatives such as pyrene butyrate, 1-pyrenesulfonyl chloride and succinimidyl 1-pyrene butyrate; QSY 7; QSY 9; reactive Red 4(

Brilliant Red 3B-A); rhodamine and derivatives, such as 6-carboxy-X-Rhodamine (ROX), 6-carboxyrhodamine (Rhodamine 6G), Rhodamine isothiocyanate, lissamine Rhodamine B sulfonyl chloride, Rhodamine B, Rhodamine 123, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red); n, N', N-tetramethyl-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. In certain embodiments, the concentration of the fluorescent probe in the compositions and methods of use is from about 0.01 μ M to about 100 μ M, from about 0.1 μ M to about 50 μ M, from about 0.1 μ M to about 10 μ M, or from about 1 μ M to about 10 μ M. In certain embodiments, the concentration of the fluorescent probe is about 0.01. mu.M, about 0.1. mu.M, about 1. mu.M, 1.1. mu.M, 1.2. mu.M, about 1.25. mu.M, about 1.3. mu.M, about 1.4. mu.M, about 1.5. mu.M, about 1.6. mu.M, about 1.7. mu.M, about 1.8. mu.M, about 1.9. mu.M, about 2. mu.M, about 2.5. mu.M, or about 5. mu.M.

Exemplary quencher labels include fluorophores, quantum dots, metal nanoparticles, and other related labels. Suitable quenching groups include Black Hole

-1 (Biosearch Technologies, Novartol, Calif.) BHQ-2, Dabcyl, Iowa

FQ (Integrated DNA Technologies, Klawille, Iowa Black RQ, QXL)^TM(Anaspec, Simon, Calif.), QSY7, QSY9, QSY21, QSY35, IRDye QC, BBQ-650, Atto540Q, Atto575Q, Atto575Q, MGB 3'CDPI3, and MGB-5' CDPI 3. In one example, the term "quenching group" refers to a substance that reduces emission from a fluorescent donor when in proximity to the donor. In preferred embodiments, the quencher group is within 1,2, 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotide bases of the fluorescent label. Fluorescence is quenched when the fluorescence emitted from the fluorophore is detectably reduced, e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. Many fluorescence quenching groups are known in the art, including Dabcyl; sulfonyl chlorides such as dansyl chloride; and Black Hole Quencher BHQ-1, BHQ-2 and BHQ-3.

In certain embodiments, an electroactive labeled probe is included in the reaction mixture and an electroactive labeled reaction product is produced. Electroactive labels that can be used as labels for generating electroactive labeled probes or oligonucleotides included in embodiments of the methods and compositions of the present application can be any of a number of electroactive labels, including but not limited to methylene blue, anthraquinone, ru (bpy)2dppz²⁺、Ru(phen)2dppz²⁺Ferrocene and derivatives thereof, hematoxylin, magnetic beads, quantum dots, biotin-advisnrp, nano-composites and the like. In certain embodiments, the concentration of electroactive probe in the compositions and methods of use is from about 0.01 μ M to about 100 μ M, from about 0.1 μ M to about 50 μ M, from about 0.1 μ M to about 10 μ M, about 1 μ M, about 1.25 μ M, about 1.5 μ M to about 10 μ M. In certain embodiments, the concentration of electroactive probe is about 0.01 μ M, about 0.1 μ M, about 1 μ M, about 1.1 μ M, about 1.2 μ M, about 1.3 μ M, about 1.4 μ M, about 1.5 μ M, about 1.6 μ M, about 1.7 μ M, about 1.8 μ M, about 1.9 μ M, about 2 μ M, about 2.5 μ M, or about 5 μ M.

Other labels may be used in the present application, including labels that allow colorimetric and chemiluminescent or fluorescent detection. For example, biotin or digoxigenin are well known in the art and can be used in combination with anti-digoxigenin antibodies and streptavidin coupled to alkaline phosphatase, horseradish peroxidase or fluorescein or rhodamine to allow colorimetric and chemiluminescent or fluorescent detection.

Any detection method or system capable of detecting a labeled nucleotide may be used in the methods according to embodiments of the present application, and such suitable detection methods and systems are well known in the art. In certain embodiments, the analyte may be detected indirectly by a cleaved probe, wherein the labeled end of the probe is released as a cleavage product. Detection of the cleavage products can be performed by methods in the group consisting of gel electrophoresis, mass spectrometry, Fluorescence Resonance Energy Transfer (FRET), lateral flow chromatography, colorimetric analysis, luminescence analysis, and electrochemical detection methods such as differential pulse voltammetry. The signal from the fluorescently labeled reaction product is detected, for example, using a photodiode.

In a preferred embodiment, the presence of the analyte is determined indirectly by means of a label cleaved from the probe. In certain embodiments, when the analyte is present, the sensor molecule no longer inhibits the restriction enzyme from recognizing the restriction enzyme site in the DNA template, and the DNA template is cleaved. The cleaved DNA template may then be hybridized to a probe, which may have a fluorescent label and a quencher group or an electrochemical label capable of ligation. In certain embodiments, the DNA template hybridized to the probe may be cleaved by one or more restriction endonucleases. This cleavage can remove the quenching group or electroactive label from the probe, open the Toehold DNA strand and expose an overhang of at least one, two, three, four, five, six, seven, eight, nine, ten, or more nucleotides in length on the unmodified DNA template strand, which serves as a nucleation site for the Toehold region and another invader probe to initiate the Toehold-mediated strand displacement reaction (TMSD).

The result of this reaction is the displacement and accumulation of the label, preferably a fragment of the invasive probe comprising a fluorescent group or an electroactive label. The newly formed hybrid invasive probe and DNA template participate in a cycling reaction of cleavage by one or more restriction enzymes followed by TMSD. Throughout the cycle, the total amount of invasive probe may be reduced, while the amount of labeled, cleaved invasive probe may be increased. Each cycle causes accumulation of displaced labeled product on the cleaved invasive probe. The signal of the label can be measured in real time or at the end point. The reaction rate is influenced by the concentration of the DNA template. The reaction rate and the final intensity of the label signal may depend on the concentration of the invasive probe.

Detection of the cleaved marker can be performed using a variety of well-known methods. Examples of detection methods include electroactive assays, fluorescent assays, or lateral flow assays. Upon restriction endonuclease cleavage, a fluorophore or an electroactive label may be released. Examples of the use of electroactive labeled probes and methods of detection using the probes are described in the art, for example, in U.S. patent No.8,975,025, which is incorporated herein by reference in its entirety.

In a lateral flow assay, for example, a sample may flow across a surface having a recognition element comprising a nucleic acid or, preferably, an antibody. The lateral flow assay may be a sandwich assay or a competitive assay. In certain embodiments, the cleaved label due to the presence of the target nucleotide may bind to the antibody-modified nanoparticle, flow through the control band, and terminate at the detection band (test band). Thus, a detection zone will appear when the target is present. When no analyte is present, the probe is not cleaved and the probe will bind to the antibody-conjugated nanoparticle and be captured by the control band.

Sensor molecules

In certain embodiments, the sensor molecule is an allosteric protein or an aptamer. The sensor molecule may be any natural or engineered protein having allosteric behavior and comprising at least one effector binding domain and at least one nucleic acid binding domain. In certain embodiments, the sensor molecule can be a monomer or a multimer. The effector binding domain can bind to the target analyte and inhibit binding of the nucleic acid binding domain to the DNA template. An aptamer can be any nucleic acid sequence that has at least one analyte binding domain and is complementary to at least 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more base pairs in a DNA template. Gaps, substitutions, deletions or other nucleic acid modifications may be present so long as the aptamer can hybridize to the DNA template. At least 1,2, 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acids may be substituted, deleted, or may be in a gap region of a complementary region. In certain embodiments, the sensor molecule may be encoded by a vector such as a plasmid vector, cosmid vector, bacterial vector, or phage vector; all of these vectors contain recombinant polynucleotides that express the polypeptides. The expressed protein may contain a tag to facilitate downstream purification. Tags include, but are not limited to, polyhistidine tags, FLAG tags, GST tags, and Myc tags. Additional steps may be required to remove the tag from the protein and are well known in the art.

In certain embodiments, the sensor molecule may be an allosteric transcription factor (aTF), such as eukaryotic aTF or prokaryotic aTF. In various embodiments, the sensor molecule can be, for example, a member of the AraC/Xlys family, ArgR family, ArsR/SmtB family, AsnC/Lrp family, Crp/Fnr family, deoR family, DtxR family, Fur family, GntR family, IcR family, LacI family, LuxR family, LysR family, MarR family, MerR family, MetJ family, ModE family, PadR family, TetR family, Xre family. Examples of the TetR family include, but are not limited to, TetR, MphR, NemR, PaaR, SaaR, RutR, SczA, RolR, Qdor, PsbI, PmeR, CymR, ComR, BetI, and TtgR. The sensor molecule may be naturally occurring (wild type) or engineered aTF.

Additional or alternative exemplary aTF are found in Microbiology and Molecular Biology Reviews by Ramos et al, 6.2005, 356. 326-; 68(3): 474-500, which are herein incorporated by reference in their entirety.

In certain embodiments, one, two, three, four, five, six, seven or more aptamers may be used as sensor molecules. In certain embodiments, provided herein are sensors for detecting the presence of a target analyte comprising an aptamer probe having a region that can bind to the analyte. The aptamers of the present application are preferably specific for a particular analyte. Aptamers can have diagnostic, target validation, and therapeutic applications. Specificity of binding is defined in terms of the dissociation constant Kd of an aptamer to its ligand. Aptamers can have high affinity with Kd range similar to antibodies (pM to nM) and specificity similar/superior to antibodies. Aptamers are typically between about 10 and about 100 nucleotides in length. For example, aptamers configured to bind to a particular target analyte can be selected by synthesizing an initial heterogeneous population of oligonucleotides and then selecting oligonucleotides within the population that bind tightly to the particular target analyte. Such aptamers may be currently existing in the art or currently commercially used, or may be developed by techniques currently commonly used in the immunological arts. Aptamer sequences are well known in the art. Aptamers can be chemically synthesized by commercial suppliers. Likewise, aptamers may be purchased from companies that develop aptamers. In addition, ligands evolved by the exponential enrichment (SELEX) system can be used to generate their own aptamers, which is an in vitro selection method well known in the art. Once an aptamer that binds to a particular target analyte is identified, the aptamer may be replicated using a variety of techniques known in biology and other arts, such as amplification by cloning or chemical synthesis and Polymerase Chain Reaction (PCR), followed by transcription or in vitro transcription.

In certain embodiments, aptamers may be present in a composition or method at a concentration of about 1nM to about 1,000nM, about 10nM to about 500nM, about 100nM to about 500nM, about 200nM to about 300nM, about 100nM, about 200nM, about 225nM, about 250nM, about 275nM, about 300nM, or about 400 nM.

Reagent kit

In certain embodiments, the compositions and methods of use of the present application may further be provided in the form of a kit. The kit may include one or more of: one or more restriction enzymes, one or more DNA templates, one or more sensor molecules, one or more invader probes, and other reagents (e.g., any of the reagents described herein, such as enzymes, buffers, or enhancers), particularly reagents that one of skill in the art would consider necessary or beneficial for a Toehold-mediated strand displacement reaction, as well as instructions for use (e.g., instructions for use including any of the methods described herein). The components of the kit may be packaged separately or together. In one example, the components are packaged together to allow for a single chamber or single tube reaction.

Enzyme

In certain embodiments, one or more enzymes, including a plurality of endonucleases, may be used. The restriction enzyme may create an overhang at the 3 'end or 5' end of the DNA template that is at least three, four, five, six, seven or eight nucleotides in length. In certain embodiments, the restriction enzyme may be a monomer or a multimer. In a preferred embodiment, one or more type IIS endonucleases can be used. Exemplary type IIS endonucleases include AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbsI, AcuHPI, AcuI, AjuI, AuI, and AuI,

BbvI、BccI、BceAI、BcgI、BciVI、BcoDI、BfuAI、BfuI、BmrI、BmsI、BmuI、BpiI、BpmI、BpuEI、

BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseMI, BsePI, BseRI, BseXI, BsgI, BslFI, BsmmAI, BsmBI-v2, BsmFI, BsmI, Bso31I, BspNI, BspMI, BspPI, BspQI, BspNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BsgBtZI, BtsCI, BstsI-v 2, BstIMuti, BveI, SaeI, CspCI, Eam I, EarI, HqqqI, Eco31I, Eco57I, Esp NI 45, FakuI, Sambi, FombeI, Fompwei, FomIII, TsIII, PpoIII, PcsPpoIi, PcsPcsPcsIII, PpoIi, PcsPcsPcsPpoIp III, PcsPqI, PqI, PcsIII, PcsPqI, PqIII, PqI, PcIII, PqI, PqIII, PcIII, PcTspGWI; or isoenzymes homologous to the restriction endonuclease thereof.

In certain embodiments, the concentration of the one or more enzymes may be from about 1U/mL to about 1000U/mL, from about 20U/mL to about 500U/mL, from about 50U/mL to about 250U/mL, or about 100U/mL.

Buffers, co-factors, metals, proteins and salts

Buffers and salts useful herein provide suitable stable pH and ionic conditions for nucleotide hybridization, Toehold-strand mediated displacement, restriction endonuclease cleavage and/or allosteric transcription factor folding, and binding to DNA templates. A variety of buffers and salt solutions and modified buffers are known in the art to be useful in the present application, including reagents not specifically disclosed herein. Preferred buffers include, but are not limited to, Tris-HCl, NaCl, MgCl₂And BSA. Preferred salt solutions include, but are not limited to, solutions of potassium acetate, potassium sulfate, potassium chloride, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, and lithium acetate.

The buffer may be present in any concentration. In some embodiments, the buffer is present in an amount from about 0.01nM to about 400mM, from about 0.05nM to about 200mM, or from about 0.1nM to about 100mM or about 50 nM. One skilled in the art will appreciate that other concentrations of buffer can be used in the present application.

In certain embodiments, the compositions and methods may further comprise coenzymes, metals, and proteins that allow for the function, stability, and folding of restriction enzymes and/or allosteric proteins.

Application method

In certain embodiments, endonucleases (restriction endonucleases), DNA templates, sensor molecules, and intrusion probes may be used to detect any target analyte in a sample. The analyte may be a small molecule, metabolite or precursor thereof, such as an antibiotic, aromatic compound, quorum sensing molecule, or metal. In a preferred embodiment, the analyte may be a metal or cation thereof, such As Hg, Cu, Ag, Au, Zn, As, Ni, Co, Cd, Pb, Fe, Ni, Mn, Cd, radicalsHg in the above-mentioned metals²⁺、Cu⁺、Ag⁺、Au⁺、Zn²⁺、As³⁺、Ni²⁺、Co²⁺、Cd²⁺、Pb²⁺、Fe²⁺、Ni²⁺、Mn²⁺And Cd²⁺(ii) a Aromatic molecules such as benzoate, n-toluate, toluene, xylene, chlorinated phenol, p-toluenesulfonate, pentachlorophenol, trichlorophenol, salicylate, 2-chloro-cis and cis-muconate; antibiotics, such as tetracycline, macrolide, chloramphenicol, actinorhodin, Ethionamide (Ethionamide) potentiator, simycin (simocyclinone), kanamycin, streptomycin, and nalidixic acid; and other small molecules such as metabolites, toxins, ester-derived molecules, hormones, and pesticides.

In particular, the compositions and methods enable the sensor molecule to control cleavage of nucleic acid sequences. The recognition capability of the sensor molecule enables a substance (particularly aTF or an aptamer) to trigger a simpler, more affordable, fast, modular and programmable nucleic acid-based loop reaction. In certain embodiments, a signal conversion and amplification circuit is provided that is independently operated, relying on the circular cleavage of a DNA template by an endonuclease. This cleavage creates a region of Toehold on the template, which is then used as a nucleation site for invasive probe hybridization. The invader probe then initiates a strand displacement reaction that ends with displacement of one of the original template strands. Importantly, this newly formed double-stranded fragment contains the DNA recognition site for the endonuclease. Thus, the fragment can be cleaved and the Toehold region formed again. The kinetics of the cycling reaction at a constant concentration of endonuclease depends on the concentration of the DNA template and the concentration and sequence of the invader probe. The invasive probe may be chemically modified with a chemical label to allow direct detection of the cleavage products by colorimetric, fluorescent or electrochemical means.

The driving of the cycling reaction may depend on the accessibility of the endonuclease to its DNA recognition site. In certain embodiments, the compositions and methods can be used to prevent access of an endonuclease to its recognition site in the absence of a cognate ligand for the sensor biomolecule.

In preferred embodiments, compositions and methods may be designed using aTF or aptamers as sensor molecules. aTF can compete with the endonuclease for the same, overlapping or adjacent DNA sequences. Aptamers can make the restriction sites unrecognized by restriction enzymes. In the absence of ligand, aTF or the aptamer inhibited DNA endonuclease cleavage. When the ligand is present and binds to aTF, aTF undergoes a conformational change and dissociates from the DNA, enabling the endonuclease to cleave and initiate a signal amplification cycle. When the ligand is present and binds to the aptamer, the aptamer may be released from the DNA template, and the DNA template may hybridize to the invasive probe. The formation of this product allows the restriction endonuclease to recognize a restriction site in the DNA template, thereby enabling the endonuclease to cut and initiate a signal amplification cycle.

In certain embodiments, the compositions and methods can be designed to transform the secondary structure of a DNA template in an aptamer. Dissociation upon binding of an aptamer to its ligand can result in a strand displacement reaction that generates a double-stranded recognition site for the endonuclease that would otherwise be "buried" in the loop structure. In certain embodiments, a restriction enzyme, such as HgaI, binds to a restriction enzyme recognition site in a double-stranded DNA molecule. A DNA template is a structure that contains the sequence complementary to the aptamer adjacent to the hairpin (i.e., stem-loop). The restriction enzyme recognition site may be buried in a loop region of the template, which is single-stranded, and thus inaccessible to the restriction enzyme. When a cognate ligand binds to the aptamer, the aptamer may dissociate from the DNA template sequence. The DNA template sequence may then hybridize to the invader probe at the aptamer complement of the template (i.e., the invader probe and aptamer may have complementary base pairs). The perfect complementarity between the template and the invasive probe allows the opening of the stem of the template and the formation of a double-stranded intermediate (S-IP) containing the recognition site for the restriction enzyme, at which point the restriction enzyme is accessible. From this point on, a cyclic TMSD and cleavage reaction occurs as previously described.

An advantageous property of the present application is that type IIS endonucleases can be utilized which function to transduce sensor/ligand binding and maintain strand displacement reactions of signal amplification. In addition, the type IIS endonuclease used in this application is capable of cleaving double-stranded DNA base pairs downstream of its recognition site. Importantly, the sequence between the recognition site and the cleavage site can be any sequence, meaning that the present application can be used with virtually any aTF or aptamer without requiring overlapping sequences between the recognition site of the endonuclease and the operator sequence, such as the DNA binding sequence of aTF or an aptamer. In addition, since the cleavage generates a Toehold region in the DNA, we used a labeled probe that performs a strand displacement reaction, so that it is successively cleaved by the endonuclease and displaced by the probe already present in the system. This enables simple, one-pot, real-time detection of small molecules.

The Toehold-mediated strand displacement-based reaction of the present application can be driven by cognate ligands (analytes) of the sensor molecule, in particular aTF and aptamers. The sensor molecules have a quantitative response to the ligand in different dynamic ranges and can be integrated in a one-pot assay, making the method simple and fast. Furthermore, the method is highly modular and programmable, since one can design biosensors for different small molecules under the same principle. Finally, sensitivity and kinetics can be fine-tuned by adjusting the concentration of the sensor molecules, the concentration and sequence of the template DNA and the invader probe.

In certain embodiments, the nucleic acid cycle of digestion with successive restriction enzymes and hybridization of the invasion probe to the template may be terminated, for example, by inactivating the restriction enzyme (e.g., by temperature change, addition of salt or other enzymes, changing pH until the restriction enzyme is inactivated), by blocking the restriction enzyme recognition site (e.g., by addition of a competing DNA strand that hybridizes to the invasion probe or template), or when there is no remaining uncleaved invasion probe.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, so long as they do not contradict the explicit teachings of the present specification.

The following are examples illustrating the practice of the methods of the present application. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are weight percentages and all solvent mixing ratios are volume ratios.

Example 1-endpoint detection of Tetracycline in Water

In the examples, the protocol provided in FIG. 1 was used to detect the antibiotic tetracycline (Tc) incorporated in a water sample by fluorescence signal.

The first step in carrying out the method is to determine the binding sequence of the ligand in response to the allosteric protein. In this example, tetracycline detection was achieved by TetR, a protein that binds tetracycline and binds to the DNA sequence TetO₁And TetO₂Conjugated natural aTF. Thus, the DNA fragment S will be bound by HgaI sequence immediately followed by TetO₁And (4) forming. A double strand S may be formed by recombining two complementary oligonucleotides. Second, the sequence of probe IP should consist of the complement of the cleaved strand containing the overhang. In this case, IP is labeled with a fluorophore FAM at the 5 'end and with a quencher BHQ-1 at the 3' end.

The DNA sequences used in this example are shown in Table 1:

TABLE 1

The TetR sequence was inserted into plasmid pET-28a and expressed in e.coli BL21 DE 3. Purification and concentration were performed by His-tag affinity chromatography and centrifugation, respectively.

The recognition sequence of HgaI is underlined, and the TetR DNA binding sequence (TetO1) is in bold.

In this example, purification was performed by recombinant expression in E.coli and using affinity chromatographyThus, TetR was obtained. Thereafter, it is necessary to measure the HgaI-to-TetO inhibition₁aTF concentration required for cleavage and subsequent Toehold-mediated strand displacement reaction. The concentration of aTF can be varied according to aTF affinity for DNA and the binding constant. In this example, titration of S by TetR showed that a 2-fold excess was sufficient to inhibit > 98% of the total reaction (fig. 7B). The titration was performed as shown in the following formulation, where the sample was replaced with ultrapure water and the final concentration of TetR varied in the range of 50nM to 500 nM.

The reaction recipe is shown in table 2:

TABLE 2

Components	Concentration of stock solution	Volume (μ L)	Final concentration
				S	1μM	0.5	50nM
TetR dimer (aTF)	2.5μM	1	250nM
				IP	10μM	1.25	1.25μM
Water (W)	-	To 10	-
				NEBuffer 1.1	10×	1	1×
Sample (I)	-	0-5.75	-
				HgaI	2000U/mL	0.5	100U/mL
Finally, the product is processed	-	10	-

According to the formulations shown in the table above, reactions were induced at 37 ℃ with different concentrations of tetracycline in the range of 25nM to 500nM, resulting in different fluorescence intensities (FIG. 8).

It should be noted that since the recognition site for HgaI is spaced from the aTF binding sequence, and the cleavage sites on the 3 'and 5' strands, 5 and 10 nucleotides downstream, respectively, are not limited by any particular sequence, this method can be used with almost any allosteric protein or aTF/ligand. In this case, only the allosteric/aTF and its cognate binding DNA sequence specific for the protein in S and IP need be altered.

Typical analysis of small molecules using the methods, portions and compositions provided herein can include preparing reactants according to the formulations in the above table, then incubating at the working temperature of the restriction enzyme and measuring the signal.

Example 2 Signal transduction and amplification circuits

The present embodiment shows the mechanism of the independent loop (fig. 2). In this example, the type IIS restriction enzyme used was HgaI, which recognizes an asymmetric DNA sequence, cleaves outside the HgaI recognition site, and generates a 5 nucleotide long Toehold starting 5 base pairs downstream of the HgaI recognition site at the 5' end of the double-stranded fragment. The Invader Probe (IP) used is an oligonucleotide perfectly complementary to the strand of the DNA template that generates the Toehold region. IP includes an HgaI recognition site near its 5' end. IP is chemically modified at the 5 'end and the 3' end respectively by a fluorescent group 6-carboxyfluorescein (6-FAM) and a quenching group Black Hole Quencher 1 (BHQ-1). The DNA template (S-IP) in this example is a double-stranded DNA fragment consisting of an unmodified strand hybridized with IP. It may also be an unmodified double-stranded DNA fragment comprising an HgaI recognition site.

In a system with HgaI, DNA template and IP (under appropriate reaction conditions); HgaI binds to its recognition site and cleaves the DNA template. This cleavage releases the BHQ-1 label from the IP and creates a five nucleotide long 5' overhang on the unmodified DNA strand that serves as both a Toehold region and a nucleation site for the Invasion Probe (IP) to initiate a Toehold-mediated strand displacement reaction (TMSD). The result of this reaction is the displacement and accumulation of P, a fluorophore-containing fragment of IP. The newly formed S-IP participates in the cyclic reaction of cleavage by HgaI and TMSD. The total amount of IP decreases and P increases throughout the cycle. Each cycle causes the accumulation of displaced fluorescent product P, whose fluorescence signal can be measured across time or endpoint. The reaction rate was influenced by the concentration of the DNA template (FIG. 3). Likewise, the reaction rate and the final fluorescence intensity depend on the concentration of IP (fig. 4).

Example 3 biosensor based on aTF

This example shows the regulation (inhibition and de-inhibition) of the loop using allosteric transcription factors and their cognate ligands (figure 1). In this example, the type IIS restriction enzyme used was HgaI, which recognizes an asymmetric DNA sequence, cleaves outside the HgaI recognition site, and generates a 5 nucleotide long Toehold starting 5 base pairs downstream of the HgaI recognition site at the 5' end of the double-stranded fragment. The DNA template (S) is a double-stranded unmodified DNA fragment comprising an HgaI recognition site immediately upstream of the operator sequence. The Invader Probe (IP) used is an oligonucleotide perfectly complementary to the strand of the DNA template that generates the Toehold region. IP includes an HgaI recognition site near its 5' end. IP is chemically modified at the 5 'end and the 3' end respectively by a fluorescent group 6-carboxyfluorescein (6-FAM) and a quenching group Black Hole Quencher 1 (BHQ-1).

This example considers a system consisting of ligands for S, IP, HgaI, aTF, and aTF. In the absence of the cognate ligand of aTF, aTF binds to the operator sequence in S and inhibits HgaI cleavage of the DNA template. If present, the ligand binds to aTF and aTF undergoes a conformational change, which reduces the affinity of aTF for the operon, causing aTF to dissociate from template S. This allows HgaI to bind to the HgaI recognition site in S, cleaving the operon sequence, and producing a product that lacks the complete operon sequence and comprises the Toehold region. IP binds to the Toehold region and initiates the TMSD reaction, the product of which is S-IP. Then, as described in example 2, a signal amplification cycle can occur.

Herein, we propose two biosensors constructed using the above mechanism. As shown in FIGS. 7A-7B, aTF TetR (SEQ ID NO:21) can be used to construct tetracycline biosensors using its corresponding operator sequence in a DNA template and partially in IP (SEQ ID NO: 19). Increasing the concentration of TetR decreases the reaction rate because HgaI has less access to DNA template. When the system is inhibited by a given concentration of TetR, the system can be de-inhibited by the ligand tetracycline. The reaction rate is proportional to the tetracycline concentration over a range of concentrations (fig. 8). Similarly, loops using the aTF MphR (SEQ ID NO:22) and its corresponding operator sequences (SEQ ID NO:17 and 18) in DNA templates and partially in IP (SEQ ID NO:20) can be used to sense macrolides. Increasing the concentration of MphR caused a decrease in the rate of reaction when erythromycin (a macrolide molecule) was not present (fig. 9A-9B). At a fixed MphR concentration, the addition of erythromycin resulted in a proportional increase in the reaction rate over a certain dynamic range (FIG. 10).

Example 4 aptamer sensor based on structural switch

Our circuit can be integrated into aptamer sensors by using different forms of structural switching mechanisms. Fig. 11A shows a scheme using a DNA template (S) with a hairpin structure comprising: 1) the HgaI recognition sequence in the loop region and 2) a single-stranded domain complementary to the ligand-responsive aptamer. The biosensor consists of a template S hybridized with an aptamer, a labeled probe (5 'and 3' ends modified with a fluorophore/quencher pair, respectively), and a type IIS restriction enzyme, which may be, but is not limited to, HgaI. The function of the aptamer is twofold: 1) inhibit hybridization of the DNA template to IP, and 2) bind its ligand with high specificity. In the presence of the ligand, the aptamer will dissociate from S and the IP can hybridize to S, thereby disrupting the hairpin and forming a double-stranded product (S-IP). The HgaI can then undergo binding and cleavage, and the TMSD reaction can proceed as previously described. Similarly, fig. 11B shows a biosensor constructed using an aptamer switch, where a hairpin structure comprising an HgaI recognition site is located downstream of the aptamer sequence. The single stranded region of S is complementary to a region of the aptamer. Similarly, the presence of a ligand in the system causes dissociation of the double stranded region between the S region and the aptamer region, so that IP can bind to S. Once a double-stranded HgaI recognition site is formed, HgaI cleaves the aptamer/ligand complex, thereby creating a Toehold region. Then, a loop as described previously can be implemented.

The stability of the hairpin stem in the DNA template S and the S region in the aptamer switch has a direct effect on the reaction leakage, reaction speed and final concentration of cleavage products. Stability can be altered by changing the length of the stem, which has a direct effect on the minimum free energy of the DNA template (fig. 12A). In a system with template S, aptamer, IP and HgaI (under appropriate reaction conditions), there is higher leakage of template S with a Short Hairpin (SH) than template S with a Long Hairpin (LH) when hybridized to the aptamer strand by 12 base pairs. Moreover, when no aptamer was present, the response was therefore not inhibited, and the system with LH was slower than the system with SH (fig. 12B).

Example 5 Signal amplification Using a lateral flow assay format

In addition to fluorescence-based detection, other formats for signal readout may be used. In this example, unlike fluorescence-based detection, IP is modified with chemical labels at the 5 'and 3' ends. These modifications may be, but are not limited to, 6-FAM, biotin, digoxin, and the like. The biosensor mechanism may be any of those described above. Except that the cleaved and displaced DNA products cannot be detected by real-time fluorescence measurements. Alternatively, the solution is added to the sample pad of the lateral flow strip test after a certain reaction time. FIG. 13 shows an example of modification of the probe with 6-FAM and biotin at the 5 'and 3' ends, respectively. The lateral flow chromatography test strip comprises: gold nanoparticles (AuNP) functionalized with anti-FAM antibodies on the sample pad (S), immobilized streptavidin on the control zone (C), and immobilized capture antibodies on the detection zone (T). After the reaction, the solution was added to the sample pad of the test strip and allowed to flow through different portions of the sample pad. In a negative assay, the 6-FAM label of the uncleaved probe binds to the anti-FAM functionalized aunps and the biotin molecule is captured by the immobilized streptavidin of the C-region, which causes aggregation of the aunps and shows a C-band. This inhibits further flow of the probe. In contrast, in the positive test, only the probe fragment containing the 6-FAM label could bind to AuNP via the anti-FAM antibody, whereas the probe fragment containing only the biotin label could not bind to AuNP. The probe fragment containing only the biotin label will be captured in the C-region by immobilized streptavidin and the band will become apparent due to AuNP aggregation. However, the probe fragment containing the 6-FAM label will continue to flow until it is captured by the capture antibody in the T-region. Therefore, the band in the T region will become apparent due to aggregation of aunps. It should be noted that this format can be designed to display a detection zone in the presence or absence of the target analyte, depending on the configuration of the test strip. Likewise, depending on the test strip, the detection and control bands may be observed in the visible spectrum or in a particular wavelength range (e.g., UV).

Example 6 Signal amplification Using an electrochemical reader

This example shows that the loop signal of the biosensor can be detected by an electrochemical method (fig. 14). The IP is labeled with an electroactive label (e.g., methylene blue) instead of a fluorophore/quencher pair, while keeping the biosensor mechanism the same as described above. Following induction of the ligand reaction, the cleavage product released is a 5 nucleotide long single-stranded DNA fragment with an electroactive label. The short segment has less negative charge and molecular weight than the all-IP, and thus can be more easily diffused to the surface of the screen printing carbon electrode, and produces a current change that can be measured using a potentiometer.

Materials and methods

Coli (e.coli) BL21 DE3 (Sangon, china) was used for recombinant protein expression. The growth medium used was Lysogenic (LB) agar or broth supplemented with kanamycin (Thermo Fisher Scientific # J17924, Waltham, Mass.) (50. mu.g/mL).

pET-28a plasmid encoding aTF was purchased directly from Sangon Biotech (Shanghai, China). This plasmid was designed to express a protein with a His-tag at the C-terminus (table 3). In Table 3, the HgaI recognition sites are underlined; spacer sequence lowercase between the HgaI binding site and the operator sequence; operon sequences are highlighted in bold; FAM is 6-carboxyfluorescein; and BHQ is Black Hole Quencher. DNA oligonucleotides forming dsDNA sensors and DNA fluorescent probes (5'6-FAM-3' BHQ1) were purchased from Integrated DNA Technologies (Clarvier, Iowa) (Table 3). The complementary oligonucleotides were recombined in TrisEDTA (TE) buffer, pH 7.5, 12.5mM MgCl, using a thermal cycler (95 ℃ 5min, -0.5 ℃/min, 20 ℃ 10min)₂The final concentration was 10. mu.M dsDNA. Accordingly, it was diluted with TE buffer pH 7.5 to the final working concentration.

TABLE 3

aTF expression and purification streaking on LB agar supplemented with kanamycin was used to pick a single colony of E.coli BL21 DE3 containing pET-28 a. A single colony was used to grow 10mL of an overnight culture at 200rpm and 37 ℃ followed by 500mL of culture. At an OD600 of 0.5, the cultures were induced with 250. mu.M IPTG (Sigma-Aldrich # I6758, St. Louis, Mo.) and grown at 200rpm for 5 hours at 37 ℃. After centrifugation at 3,300 Xg for 20 minutes, the cell pellet was recovered and resuspended in lysis buffer (PBS pH 7.4(Thermo Fisher Scientific #28372), 1 XHart protease inhibitor (Thermo Fisher Scientific #1861278), 10mM imidazole) or stored at-20 ℃ for up to 7 days in PBS pH 7.4. The suspension was sonicated on ice (1min sonication for 1min stationary, 5 cycles with a duty cycle of 50%) and centrifuged at 13,000 × g for 30 min. The supernatant was then used to purify the His-tagged protein using Ni-NTA agarose (Qiagen #30210 of hilden, germany) in a gravity column (10mM, 25mM and 250mM imidazole in PBS for equilibration, washing and elution, respectively). Desalting (final buffer PBS) was performed using a centrifugal filter (Amicon Ultra4, Millipore of burlington, massachusetts) and the purified protein was concentrated. Purification was confirmed by reduced SDS-PAGE 12% (FIG. 15), and concentration was measured by Coomassie Brilliant blue Assay (Bradford Assay). The protein was kept in 50% glycerol at-20 ℃ and diluted accordingly with PBS.

Gel electrophoresis and electrophoretic mobility shift analysis HgaI activity on DNA templates with different length spacers (SEQ ID NO:2 to SEQ ID NO:16) was observed using PAGE 12% (room temperature, 90V, TBE 1 Xbuffer) (FIG. 5B). The reaction containing the DNA template, TetR and HgaI was incubated at 37 ℃ and terminated after 30 minutes by addition of proteinase K (New England Biolabs # P8107S, Ipsweck, Mass.) after an additional 10 minutes. This step was to terminate the HgaI and release any uncleaved DNA from the TetR.

To evaluate the allosteric effect of TetR (fig. 5A and 5C), dsDNA was mixed with aTF in different ratios and equilibrated in nebuffer1.1(New England Biolabs # B7201S) at room temperature. EMSA was performed on 10% PAGE in 90V, TBE 1 Xbuffer at room temperature. Post-staining was performed with SYBRgold (Thermo Fisher Scientific # S11494)1X in TBE 1X buffer.

The reaction compositions are shown in the respective figures, and all reactions are carried out using a final concentration of 1 × NEBuffer1.1(New England Biolabs # B7201S) unless otherwise indicated.

The reaction was constructed by adding buffer1.1(New England Biolabs), dsDNA template, invasion probe, aTF, nuclease-free water (Integrated DNA Technologies) or sample and Hgal (New England Biolabs) to a final volume of 10. mu.L. Immediately after the addition of HgaI, the 6-FAM fluorescence intensity was monitored for 30 to 120 minutes on channel 1 of the CFX96 Touch Real Time PCR detection System (Bio-Rad Laboratories, Inc., Heracles, Calif.). The detailed formulation of the Hgal restriction reaction can be found in Table 4.

TABLE 4

Components	Concentration of stock solution	Volume (μ L)	Final concentration
				dsDNA template	1μM	0.5	50nM
aTF	Multiple kinds of	1.75	Multiple kinds of
				Probe needle	10μM	1.25	1.25μM
Water or sample	-	5	-
				Buffer 1.1NEB	10X		1	1X
Hga1	2000U/mL	0.5	100U/mL
				Final	-	10	-

Water sampling and treatment water samples were collected from ponds (22.332022, 114.245774) in the east of hong kong. Samples were collected in sterile 50mL Falcon tubes and transported to the laboratory at room temperature (1 h). The water samples were filtered in the laboratory using a 0.45 μm syringe filter and used immediately for the reaction. Prior to filtration, known concentrations of tetracycline (see SigmaACS Synthetic Biology pubs. ACS. org/synthtbio Research articule https:// dx. doi. org/10.1021/acssynbio.0c00545 ACS Synth. biol.2021,10, 371. 378376 Sigma-Aldrich #28 871), erythromycin (Sangon Biotech CAS #114-07-8), kanamycin (Thermo Fisher Scientific # J17924) or ampicillin (Sigma-Aldrich # A9518) were incorporated into the corresponding samples. All reactions with water samples (spiked or not) were performed on the day of collection with a maximum of 2h between collection and reaction. For the data shown in FIG. 6C, a TetR-based biosensor was detected using 500nM (final concentration of 250nM) antibiotic and 2.5 μ M (final concentration of 1.25nM) was used for the MphR-based biosensor. In all reactions, 50nM DNA template and 1.25. mu.M invader probe were used (FIGS. 6A-6C).

All raw data were processed and analyzed using Microsoft Excel and GraphPad Prism 8. All plots were drawn with GraphPad Prism 8 (Prism in san diego, california) and Matlab (Mathworks in botra valley, california). Com, by biorender.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Furthermore, any element or limitation of any invention or embodiment thereof disclosed herein may be combined with any and/or all other elements or limitations (alone or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are included within the scope of the invention, but are not limited thereto.

SEQUENCE LISTING

<110> hong Kong university of science and technology

<120> detection of analytes by enzyme-mediated strand displacement reaction

<130> HKUS.160X

<150> 63/103,492

<151> 2020-08-06

<160> 22

<170> PatentIn version 3.5

<210> 1

<211> 642

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 1

atgagtcggt tagacaagag taaagtgatt aattcggctc tcgaactgct gaatgaagtt 60

gggattgagg ggttgactac ccgcaaatta gcacagaaac ttggcgtaga acagccaact 120

ctttactggc acgttaagaa taagcgggcc cttcttgatg cgcttgccat cgagatgctg 180

gaccgccatc acacacactt ttgcccatta gaaggggagt cgtggcagga tttcttacgg 240

aataatgcca agtctttccg gtgcgctctt cttagccatc gtgacggtgc aaaggtacat 300

ttaggcacgc gcccgaccga aaaacagtac gaaaccttag aaaaccagct tgcctttctg 360

tgtcaacagg gtttcagcct cgaaaatgcg ttatacgctc tgtcggccgt aggccacttt 420

acgctcgggt gcgtcctcga ggaccaagag caccaggtcg ctaaggagga gcgggagacc 480

ccaaccacag atagtatgcc accattgtta cgtcaagcaa tcgagttgtt tgatcaccaa 540

ggtgcggagc ctgcatttct ttttggttta gaactgatta tctgtggcct tgaaaagcag 600

ttgaaatgcg aaagcgggtc ctgacatcat catcatcatc at 642

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ataaagacgc tccctatcag tgatagaga 29

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ataaagacgc tccctatcag tga 23

<210> 5

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<223> DNA template sequence for use with TetR sensor molecules

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ataaagacgc agtccctatc agtgatagag a 31

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<223> DNA template sequence for use with TetR sensor molecules

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tctctatcac tgatagggac tgcgtcttta t 31

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<212> DNA

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<223> DNA template sequence for use with TetR sensor molecules

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ataaagacgc agtctcccta tcagtgatag aga 33

<210> 8

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<223> DNA template sequence for use with TetR sensor molecules

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<223> DNA template sequence for use with TetR sensor molecules

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<213> Artificial Sequence (Artificial Sequence)

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<223> DNA template sequence for use with TetR sensor molecules

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<223> DNA template sequence for use with TetR sensor molecules

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<213> Artificial Sequence (Artificial Sequence)

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<223> DNA template sequence for use with TetR sensor molecules

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ataaagacgc agtcagtcag tccctatcag tgatagaga 39

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<223> DNA template sequence for use with TetR sensor molecules

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tctctatcac tgatagggac tgactgactg cgtctttat 39

<210> 15

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<223> DNA template sequence for use with TetR sensor molecules

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ataaagacgc agtcagtcag tctccctatc agtgatagag a 41

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<223> DNA template sequence for use with TetR sensor molecules

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<213> Artificial Sequence (Artificial Sequence)

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<223> DNA template sequences for use with MphR sensor molecules

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ataaagacgc gaatataacc gacgtgactg ttacatttag gtgg 44

<210> 18

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> DNA template sequences for use with MphR sensor molecules

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ccacctaaat gtaacagtca cgtcggttat attcgcgtct ttat 44

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<223> exemplary invasive Probe

<220>

<221> misc_feature

<222> (1)..(1)

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<222> (23)..(23)

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ataaagacgc tccctatcag tga 23

<210> 20

<211> 23

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<213> Artificial Sequence (Artificial Sequence)

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<223> exemplary invasive Probe

<220>

<221> misc_feature

<222> (1)..(1)

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<222> (23)..(23)

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ataaagacgc gaatataacc gac 23

<210> 21

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<213> Escherichia coli (Escherichia coli)

<400> 21

Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu

1 5 10 15

Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln

20 25 30

Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys

35 40 45

Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His

50 55 60

Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg

65 70 75 80

Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly

85 90 95

Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr

100 105 110

Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu

115 120 125

Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys

130 135 140

Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr

145 150 155 160

Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu

165 170 175

Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu

180 185 190

Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser

195 200 205

<210> 22

<211> 194

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 22

Met Pro Arg Pro Lys Leu Lys Ser Asp Asp Glu Val Leu Glu Ala Ala

1 5 10 15

Thr Val Val Leu Lys Arg Cys Gly Pro Ile Glu Phe Thr Leu Ser Gly

20 25 30

Val Ala Lys Glu Val Gly Leu Ser Arg Ala Ala Leu Ile Gln Arg Phe

35 40 45

Thr Asn Arg Asp Thr Leu Leu Val Arg Met Met Glu Arg Gly Val Glu

50 55 60

Gln Val Arg His Tyr Leu Asn Ala Ile Pro Ile Gly Ala Gly Pro Gln

65 70 75 80

Gly Leu Trp Glu Phe Leu Gln Val Leu Val Arg Ser Met Asn Thr Arg

85 90 95

Asn Asp Phe Ser Val Asn Tyr Leu Ile Ser Trp Tyr Glu Leu Gln Val

100 105 110

Pro Glu Leu Arg Thr Leu Ala Ile Gln Arg Asn Arg Ala Val Val Glu

115 120 125

Gly Ile Arg Lys Arg Leu Pro Pro Gly Ala Pro Ala Ala Ala Glu Leu

130 135 140

Leu Leu His Ser Val Ile Ala Gly Ala Thr Met Gln Trp Ala Val Asp

145 150 155 160

Pro Asp Gly Glu Leu Ala Asp His Val Leu Ala Gln Ile Ala Ala Ile

165 170 175

Leu Cys Leu Met Phe Pro Glu His Asp Asp Phe Gln Leu Leu Gln Ala

180 185 190

His Ala

Claims

1. A composition for the detection of an analyte comprising a restriction enzyme, a DNA template, a sensor molecule, and an invasive probe, wherein the analyte binds to the sensor molecule and allows the restriction enzyme to recognize a restriction enzyme site in the DNA template, and wherein the invasive probe in combination with a reporter molecule produces a detectable signal.

2. The composition of claim 1, wherein the sensor molecule is an allosteric transcription factor or an aptamer.

3. The composition of claim 1, wherein the analyte is a ligand that binds to the sensor molecule.

4. The composition of claim 1, wherein the restriction enzyme is a type IIS restriction enzyme that creates an overhang at the 3 'end or 5' end of the DNA template, and the overhang is at least four nucleotides in length.

5. The composition of claim 1, wherein the DNA template is a double-stranded DNA sequence or a single-stranded DNA sequence; the double-stranded DNA sequence comprises at least one restriction site upstream or downstream of at least one operator sequence, and the single-stranded DNA sequence comprises a restriction site within the loop region of the hairpin structure and a domain complementary to the aptamer molecule.

6. The composition of claim 2, wherein the DNA template sequence and the aptamer sequence are capable of being ligated in a single DNA oligonucleotide.

7. The composition of claim 6, wherein the single DNA oligonucleotide forms a hairpin structure in which the stem of the hairpin is the region of complementarity between the DNA template and the aptamer.

8. The composition of claim 1, wherein the sensor molecule binds or hybridizes to the DNA template when the analyte is not present.

9. The composition of claim 1, wherein the sensor molecule does not bind or hybridize to the DNA template when the analyte is present.

10. The composition of claim 1, wherein in the absence of the analyte, the sensor molecule does not bind or hybridize to the DNA template with the same affinity as in the presence of the analyte.

11. The composition of claim 1, wherein the invasive probe is a single-stranded DNA sequence that is partially complementary or fully complementary to a DNA template.

12. The composition of claim 1, wherein the invasive probe has a fluorescent label, a quenching label, and/or an electroactive label at the 3 'end or 5' end.

13. The composition of claim 2, wherein the allosteric transcription factor comprises at least one effector binding domain and at least one nucleic acid binding domain.

14. The composition of claim 5, wherein the allosteric transcription factor bound to the double-stranded DNA template inhibits recognition of a restriction enzyme site by the restriction enzyme.

15. The composition of claim 5, wherein the hairpin structure of the single-stranded DNA sequence inhibits recognition of the restriction enzyme site by the restriction enzyme.

16. The composition of claim 5, wherein the invasive probe hybridizes to a region of the DNA template having a single-stranded DNA sequence complementary to the aptamer, or to a stem-loop region of the DNA template.

17. The composition of claim 7, wherein the hairpin structure of the DNA template inhibits recognition of the restriction enzyme site by the restriction enzyme.

18. The composition of claim 7, wherein the invasive probe hybridizes to a region of the DNA template that is complementary to the aptamer, or to a stem-loop region of the DNA template.

19. The composition of claim 1, further comprising a buffer suitable for restriction enzyme cleavage reactions and Toehold-mediated strand displacement reactions.

20. The composition of claim 1, wherein the DNA template comprises at least one restriction site for a restriction enzyme and at least one allosteric protein binding sequence.

21. A method of detecting an analyte in a sample, the sample being used to regulate a DNA loop, the method comprising:

a) contacting the sample with the composition of claim 1; and

b) binding the sensor molecule to the DNA template; wherein the sensor molecule prevents the restriction enzyme from recognizing a restriction enzyme site in the DNA template specific for the restriction enzyme when the analyte is not present or displaces the sensor molecule from the DNA template when the analyte is present and the analyte binds to the sensor molecule.

22. The method of claim 21, wherein when the analyte is present, the method further comprises:

c) digesting the DNA template with the restriction enzyme;

d) hybridizing the invader probe to the DNA template;

e) cleaving the hybridized invader probe and DNA template with the restriction enzyme; and

f) determining the presence or absence of at least one analyte by detecting a signal released from the cleaved hybridized invader probe and DNA template, wherein detection of said signal is indicative of the presence of said analyte.

23. The method of claim 21, wherein the analyte is selected from an antibiotic, an aromatic, a quorum sensing molecule, or a metal.

24. The method of claim 21, wherein the signal is an electrochemical signal, a fluorescent signal, a luminescent signal, and/or a colorimetric signal.

25. The method of claim 21, further comprising contacting the sample with a buffer suitable for restriction enzyme cleavage reaction and a Toehold-mediated strand displacement reaction.

26. The method of claim 21, wherein the sample is treated by heating, centrifugation, chemical or physical dissolution, dilution, concentration, or filtration prior to being mixed in contact with the composition of claim 1.

27. The method of claim 21, wherein a) mixing and contacting and b) combining are performed at a temperature of about 20 ℃ to about 60 ℃.

28. The method of claim 22, wherein c) digesting, d) hybridizing and e) cleaving are performed at a temperature of about 20 ℃ to about 60 ℃.

29. The method of claim 22, wherein e) cleaving further comprises hybridizing the invader probe to the cleaved hybridized invader probe and DNA template and replacing short strands of cleaved DNA fragments.

30. A method of regulating a DNA loop, the method comprising:

a) contacting a restriction enzyme, a DNA sequence having a restriction enzyme recognition site, and an invasion probe;

b) digesting the DNA sequence with the restriction enzyme, thereby generating a digested DNA sequence having a Toehold region;

c) hybridizing said invader probe to said digested DNA sequence of step b) at said Toehold region to produce a hybridized DNA sequence;

d) digesting the hybrid DNA sequence of step c) with the restriction enzyme, thereby generating a digested DNA sequence having a second Toehold region;

e) hybridizing said invader probe to said digested DNA sequence of step d) at said second Toehold region; and

f) optionally, repeating steps d) and e) until the restriction enzyme is inactivated or there is no remaining uncleaved invasion probe or is terminated by blocking the restriction enzyme recognition site.

31. The method of claim 30, wherein the digestion of step d) releases a label or quenching group from the invasive probe.

32. The method of claim 30, wherein the hybridizing of step e) releases a label from the invasive probe.